Molecular rulers are rigid, variable-length crosslinking reagents which we are developing to probe the structure of biological macromolecules. Oligomers of proline provide the rigidity and variable lengths while different crosslinking reagents are used on the ends of different targets. The positions of ribosomal proteins in 30S and 50S subunits have been probed by a variety of techniques, including crosslinks. The 50S subunit structure is not yet as well understood as the 30S. We propose to crosslink 50S proteins to the P site by binding chlorambucilyl oligoprolyl phenylalanyl-tRNA to 70S ribosomes programmed by poly (U), and examine by polyacrylamide gel fingerprints which 50S proteins are chlorambucil alkylated for a given length of oligoproline ruler. For finer resolution, we propose to isolate crosslinked proteins from gels and separate their tryptic fragments by fingerprinting or HPLC. Fragments which are labeled due to alkylation by labeled molecular rulers may be identified by amino acid compositions, which will be determined by HPLC of fluorescently modified amino acids following hydrolysis.